Gene Editing of Mouse Embryonic and Epiblast Stem Cells

Molecular and Cellular Biology

Waardenberg

Demuth

et al. 2020

Self Cite

The extensive array of basic helix-loop-helix (bHLH) transcription factors and their combinations as dimers underpin the diversity of molecular function required for cell type specification during embryogenesis. The bHLH factor TWIST1 plays pleiotropic roles during development. However, which combinations of TWIST1 dimers are involved and what impact each dimer imposes on the gene regulation network controlled by TWIST1 remain elusive. In this work, proteomic profiling of human TWIST1-expressing cell lines and transcriptome analysis of mouse cranial mesenchyme have revealed that TWIST1 homodimers and heterodimers with TCF3, TCF4, and TCF12 E-proteins are the predominant dimer combinations. Disease-causing mutations in TWIST1 can impact dimer formation or shift the balance of different types of TWIST1 dimers in the cell, which may underpin the defective differentiation of the craniofacial mesenchyme. Functional analyses of the loss and gain of TWIST1–E-protein dimer activity have revealed previously unappreciated roles in guiding lineage differentiation of embryonic stem cells: TWIST1–E-protein heterodimers activate the differentiation of mesoderm and neural crest cells, which is accompanied by the epithelial-to-mesenchymal transition. At the same time, TWIST1 homodimers maintain the stem cells in a progenitor state and block entry to the endoderm lineage.

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

TWIST1 Homodimers and Heterodimers Orchestrate Lineage-Specific Differentiation

Molecular and Cellular Biology

Waardenberg

Demuth

et al. 2020

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“…ESCs of specific genotype (non-fluorescent) were injected into 8-cell host wildtype embryos (expressing fluorescent DsRed.t3) and chimeras were collected at E9.5 or E11.5 (Figure 3A) (Sibbritt et al, 2019).…”

Section: Genetic Interaction Of Twist1 and Chromatin Regulators In Craniofacial Morphogenesismentioning

confidence: 99%

“…ESC clones with monoallelic frameshift mutations and the parental A2LoxCre ESC line were used to generate chimeras. Embryo injections were performed as previously described (Sibbritt et al, 2019). Briefly, 8-10 ESCs were injected per eight-cell DsRed.T3 embryo (harvested at 2.5 dpc from super-ovulated ARC/s females crossed to DsRed.T3 stud males) and incubated overnight.…”

Section: Generation Of Mouse Chimeras From Escsmentioning

confidence: 99%

TWIST1 and chromatin regulatory proteins interact to guide neural crest cell differentiation

Masamsetti

Sun

et al. 2021

eLife

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Protein interaction is critical molecular regulatory activity underlining cellular functions and precise cell fate choices. Using TWIST1 BioID-proximity-labelling and network propagation analyses, we discovered and characterized a TWIST-chromatin regulatory module (TWIST1-CRM) in the neural crest cells (NCC). Combinatorial perturbation of core members of TWIST1-CRM: TWIST1, CHD7, CHD8, and WHSC1 in cell models and mouse embryos revealed that loss of the function of the regulatory module resulted in abnormal differentiation of NCCs and compromised craniofacial tissue patterning. Following NCC delamination, low level of TWIST1-CRM activity is instrumental to stabilize the early NCC signatures and migratory potential by repressing the neural stem cell programs. High level of TWIST1 module activity at later phases commits the cells to the ectomesenchyme. Our study further revealed the functional interdependency of TWIST1 and potential neurocristopathy factors in NCC development.

“…Mutant ESCs for Twist1 and the three validated NCC-exclusive chromatin regulatory partners Chd7, Chd8 and Whsc1 were generated by CRISPR-Cas9 editing (Figure S3A,B) (Ran et al, 2013). ESCs of specific genotype (non-fluorescent) were injected into 8-cell host wildtype embryos (expressing fluorescent DsRed.t3) and chimeras were collected at E9.5 or E11.5 ( Figure 3A) (Sibbritt et al, 2019). Only embryos with predominant contribution of mutant ESCs, indicated by absence or low level of DsRed.t3 fluorescence were analyzed.…”

Section: Genetic Interaction Of Twist1 and Chromatin Regulators In Crmentioning

confidence: 99%

TWIST1 and chromatin regulatory proteins interact to guide neural crest cell differentiation

Masamsetti

Sun

et al. 2020

Preprint

Self Cite

Protein interaction is critical molecular regulatory activity underlining cellular functions and precise cell fate choices. Using TWIST1 BioID-proximity-labelling and network propagation analyses, we discovered and characterized a TWIST-chromatin regulatory module (TWIST1-CRM) in the neural crest cell (NCC). Combinatorial perturbation of core members of TWIST1-CRM: TWIST1, CHD7, CHD8, and WHSC1 in cell models and mouse embryos revealed that loss of the function of the regulatory module resulted in abnormal specification of NCCs and compromised craniofacial tissue patterning. Our results showed that in the course of cranial neural crest differentiation, phasic activity of TWIST1 and the interacting chromatin regulators promote the choice of NCC fate while suppressing neural stem cell fates, and subsequently enhance ectomesenchyme potential and cell motility. We have revealed the connections between TWIST1 and potential neurocristopathy factors which are functionally interdependent in NCC specification. Moreover, the NCC module participate in the genetic circuit delineating dorsal-ventral patterning of neural progenitors in the neuroepithelium.